High serum cholesterol is commonly associated with an increased risk of heart-attack, atherosclerosis and circulatory disorders. In addition, a variety of diseases are caused by disorders of cholesterol catabolism, such as gallstone disease, atherosclerosis, hyperlipidemia and some lipid storage diseases.
The major pathway for disposal of cholesterol in the body is by secretion of cholesterol and bile acids into the gut. Bile contains free cholesterol and bile acids. The enzyme, cholesterol 7.alpha.-hydroxylase (CYP7), commits cholesterol to bile acid synthesis and catalyzes the first and rate-limiting step of bile acid synthesis in the liver hydroxylation of cholesterol at the 7.alpha.-position, thereby forming 7.alpha.-hydroxycholesterol. Thus, by increasing synthesis of bile acids, this enzyme plays a key role in the liver by depleting hepatic cholesterol pools, resulting in increased LDL uptake and a lowering of serum cholesterol levels.
Bile acids are physiological agents which are important in the solubilization of lipid-soluble vitamins, sterol and xenobiotics. Bile acids are synthesized exclusively in the liver and are secreted to the intestines where they are modified to secondary bile acids. Most bile acids are reabsorbed in the ileum and recirculated to the hepatocytes via the portal vein. The feedback of bile into the liver is known to inhibit cholesterol 7.alpha.-hydroxylase and thus inhibit the overall rate of bile acid synthesis. Cholesterol 7.alpha.-hydroxylase therefore has been a subject of intense investigations to elucidate the regulatory mechanisms of bile acid synthesis in the liver.
It is known that an interruption of bile acid reabsorption, such as that caused by the bile sequestrant, cholestyramine, or by a bile fistula, stimulates the rate of bile acid synthesis and cholesterol 7.alpha.-hydroxylase activity in the liver. It is believed that cholesterol 7.alpha.-hydroxylase activity in the liver is regulated primarily at the gene transcriptional level by bile acids, cholesterol, hormones, diurnal rhythm and other factors.
Generally, the regulation of eukaryotic genes is thought to occur at several locations, including the promoter sequences, which are located upstream of the transcription start site; enhancer or repressor sequences, which are located upstream of the promoter; within intron sequences, which are non-coding sequences located between exons or coding sequence; and in 3' sequences, which are located downstream from the coding region. The promoter sequence is unique to each gene and is required for the accurate and efficient initiation of gene transcription. Enhancers and/or repressors regulate promoter activity and determine the level of gene transcription during the development and differentiation of a particular tissue.
The promoter of most eukaryotic genes contains a canonical TATA box that binds a TFIID TATA box binding protein. TFIID complex and associated transcription activators (TAFs) interact with the basal initiation factors and RNA polymerase II to activate the promoter. The transcription complex assembly and initiation are regulated by transcription factors bound to enhancer elements located in the promoter and other regions of the gene (Pugh and Tjian, J. Biol. Chem. 267,679-682, 1992). Tissue-specific transcription factors and nuclear steroid hormone receptors are known to play an important role in the regulation of gene expression in different tissues during development and differentiation.
However, the mechanisms underlying the regulation of cholesterol 7.alpha.-hydroxylase gene expression at the molecular level are not understood. An understanding of the regulation of CYP7 gene expression would permit development of therapeutics for treating patients with defects in bile acid synthesis and cholesterol metabolism due to altered (deficient or excessive) gene expression.
In order to study the mechanism of regulation of human cholesterol 7.alpha.-hydroxylase at the molecular level, it is therefore important to determine the correct coding, non-coding and promoter region gene sequences. An elucidation of the enzyme's gene structure, a method for analyzing promoter and enhancer/repressor activity, as well as transgenic animal models with which to study human cholesterol 7.alpha.-hydroxylase, are desired. Attempts to provide a transgenic animal expressing recombinant CYP7 have not been successful using the cDNA of CYP7. Thus, important discoveries concerning the CYP7 gene and systems for studying the CYP7 enzyme's physiology, each of which aims towards the design of therapeutic drugs and the treatment of patients with defects in bile acid synthesis and cholesterol metabolism, are highly desired.
To understand the structure and function of human CYP7 and its regulation by factors, such as bile acids, cholesterol and hormones, it is essential to purify the human CYP7 enzyme. However, the CYP7 enzyme is present in an extremely low levels in human liver; therefore, it has not been possible to isolate sufficient quantities of purified, functional enzyme from human livers.
Although a cDNA molecule encoding human CYP7 enzyme has been determined, recombinant expression of human CYP7 has not heretofore been achieved. Karam and Chiang, Biochem. Biophys. Res. Commun. 185:588 (1992). Recently, a strategy to express a catalytically active, truncated rat cholesterol 7.alpha.-hydroxylase in E. coli was disclosed. Li and Chiang, J. Biol. Chem. 266 (29): 19186 (1991). The disclosures of both of those publications are expressly incorporated herein by reference. In the latter publication, it was disclosed that the expression of a membrane-bound hydrophobic protein in E. coli is difficult because the bacteria lacks internal membranes. Via PCR, a modified cDNA was generated that encoded a truncated enzyme lacking the N-terminal 23 amino acid residues of the rat cholesterol 7.alpha.-hydroxylase enzyme. The resulting protein was expressed, predominantly in the cytosol of the bacteria. The purified recombinant enzyme was active, as determined by its ability to hydroxylate cholesterol in a reconstituted system, and has a K.sub.m for cholesterol and V.sub.max similar to those of the rat microsomal (non-truncated) enzyme.
Despite the high sequence identity between the rat and human cholesterol 7.alpha.-hydroxylase, however, it previously has not been possible to express the human cholesterol 7.alpha.-hydroxylase in E. coli following the same strategy and using the same expression vector (pKK233-2) as that previously used for the expression of rat cholesterol 7.alpha.-hydroxylase. Thus, a catalytically active, recombinant human CYP7 enzyme is desirable. Recombinantly-expressed, truncated human CYP7 could be used to detect agents that stimulate or inhibit human CYP7's catalytic activity. Further, such recombinant protein can be used to produce anti-CYP7 antibodies which would be useful for screening assays, for example, to detect stimulated or inhibited production of human CYP7 in response to exposure of a compound to a human CYP7-producing culture.